3b), and it showed that

Asp409del mutant dramatically cha

3b), and it showed that

Asp409del mutant dramatically changed the Na+-binding loop conformation and the Na+-binding residues (Try185 and Asp185a) in the mutant loop were off the Na+ binding sites. Therefore, Na+ binding capacity of the loop would be impaired, and the conformation of the neighbouring catalytic pocket and FVa-binding helix (residues 163–170) might be changed. The enzymatic activity of the Asp409del mutant FXa would be affected, consistent with our in vitro expression study results. In this report, we analysed a proband with this website severe FX deficiency who has experienced abnormal bleeding since childhood. Two novel heterozygous mutations were identified in the proband: one was a splice mutation (IVS5+1G>A) and the other was an in-frame deletion of aspartic acid 409 (Asp409del), which RG-7388 ic50 is associated with the CRM+ mutation. Mutations that occur in the GT-AG consensus splice sequences are certain to result in incorrect splicing, but aberrant splicing patterns are

heterogeneous, resulting in either complete skipping of the exon, retention of the intron, or introduction of a new splice site within either an exon or intron [5]. All splice sites of the F10 gene are GT-AG consensus pairs. At present, five splice-site mutations in the F10 gene have been reported, but only two have been studied using in vitro splicing and ectopic transcript analysis [3, 6]. In this study, a donor splice-site mutation in intron 5 (IVS5+1G>A)

was studied by ectopic transcription combined with informative marker analysis. The result of ectopic transcription showed that no aberrant transcript from the IVS5+1G>A mutant allele was found. Because the proband was homozygous for Fossariinae all reported polymorphisms in the F10 gene (data not shown), the heterozygous deletion (Asp409del) in exon 8 identified in the other allele was used as an informative marker, and sequencing results confirmed the loss of the transcript from the splice-site mutant allele. Therefore, the use of an informative marker in this study provided direct evidence of the absence of an aberrant transcript from the splice-site mutant. The possible reason for the absence of such a transcript might be that the mRNA derived from the IVS5+1G>A mutant allele is unstable and degrades rapidly in vivo. Therefore, a computer-assisted analysis of splice-site prediction in the DNA sequence was performed with a program available online (http://www.fruitfly.org/seq_tools/spliceAbst.html). The program predicted that the mutation might completely abolish the physiological splice site and result in two candidate splice sites with the same splice-site score of 0.83 (physiological score of 0.94). Both transcripts originating from these two alternative splice sites were predicted to possess terminal codons at nucleotide positions 82–84 of exon 6 and 111–113 of intron 5 respectively.

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